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How to increase A260/280 and A260/230 ratio for microarray experiment? - (May/04/2009 )

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.

-wntiong-

Hey there :P

Have you tried precipitating your RNA? We have to do this for our DNA as after amplification the ratios are a bit rubbish. After precipitation it's all good ;)

Clare

wntiong on May 5 2009, 01:08 AM said:

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.

-Clare-

Clare on May 5 2009, 05:43 PM said:

Hey there :wacko:

Have you tried precipitating your RNA? We have to do this for our DNA as after amplification the ratios are a bit rubbish. After precipitation it's all good :D

Clare

wntiong on May 5 2009, 01:08 AM said:

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.



can i Re-precipitate the RNA with Isopropanol again or perform more ethanol wash? thanks

-wntiong-

can i Re-precipitate the RNA with Isopropanol again or perform more ethanol wash? thanks



For both RNA and DNA I add 2vol Abs. EtOH and NaOAc to a final conc. of 0.3M. Chuck it in -80 (either ON or at least 30 mins) or put tubes on dry ice for 5-10 mins.
Spin 30 mins, max speed (if using eppendorf tubes).
Remove SN and add 70% EtOH to wash.
Air dry pellet and resuspend in water :wacko:

Clare

-Clare-

Thanks for your reply.

Because i am dealing with rna isolation from whole blood, and often get a pink aqueous phase during rna isolation, i am not sure if this affect the rna quality.. any advise? thanks

wenni

-wntiong-

wntiong on May 7 2009, 03:33 AM said:

Thanks for your reply.

Because i am dealing with rna isolation from whole blood, and often get a pink aqueous phase during rna isolation, i am not sure if this affect the rna quality.. any advise? thanks

wenni


Do you remove the RBCs before you try and isolate RNA?
C

-Clare-

wntiong on May 4 2009, 05:08 PM said:

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.



Are you getting poor ratios even after purifying your RNA with a purification kit, like that from QIAgen??? because, i usually get much improved ratios after this purification step.

-DRN-

DRN on May 8 2009, 11:29 AM said:

wntiong on May 4 2009, 05:08 PM said:

Hi,

I often get a poor ratio of A260/280 and A260/230 ratio from several rna isolations i did.

Since i going to do microarray work, can give me some advise how to increase these ratios? Any tip?

Thank you.



Are you getting poor ratios even after purifying your RNA with a purification kit, like that from QIAgen??? because, i usually get much improved ratios after this purification step.


yeah run it through a qiagen RNeasy kit

-Ahrenhase-

Clare on May 7 2009, 04:53 PM said:

wntiong on May 7 2009, 03:33 AM said:

Thanks for your reply.

Because i am dealing with rna isolation from whole blood, and often get a pink aqueous phase during rna isolation, i am not sure if this affect the rna quality.. any advise? thanks

wenni


Do you remove the RBCs before you try and isolate RNA?
C



Hi Clare,

I didnt remove RBC, but stabilize in tri-reagent immediately after blood was drawn.

-wntiong-